5 replies to this topic

[jal27]

Hello every one

I am new to this forum
I have a difficulty with e.coli protein expression.
When i pick colony and grow them overnight, they are growing very well..but they are not growing when i start my actuall protein expression next morning.  
I have been doing this since long and i never had any problem...:
Please help me...

[rosepolaris]

It reminds me that once my colleague used a wrong antibiotic....

jal27, on 30 July 2010 - 08:22 AM, said:

Hello every one

I am new to this forum
I have a difficulty with e.coli protein expression.
When i pick colony and grow them overnight, they are growing very well..but they are not growing when i start my actuall protein expression next morning.  
I have been doing this since long and i never had any problem...:
Please help me...

[jal27]

Well I am using correct antibiotic....but somehow my cells are not growing...

[pDNA]

what antibiotic do you use?

Regards,
p

jal27, on 04 August 2010 - 06:55 AM, said:

Well I am using correct antibiotic....but somehow my cells are not growing...

[protolder]

Hola,I think that you have two problems the toxicity of your recombinant protein and the host strain. The toxicity kills your bacteria and you are using a strain that don´t repress the expression withouth inducer,as BL21 or any of its derived wich aren´t lac Iq and you have an scape in the synthesis wich is lethal. Try to use lacIq strains and add o.2 g/l of glucose to the preinoculum. In this way only the expression will do when you add inducer. Good luck

[ram]

If you have been doing expression of the same protein in the same strain and same vector and if you were getting expected growth, then there should not be any problem of toxicity. I will suggest to check EVERYTHING! There is no other reason than that you are doing something wrong. Confirm that you are really using same plasmid and strain/glycerol stock for culture initiation, same antibiotic, same media and what else?